Flow cytometry is a technology that interrogates a large number of cells flowing in a stream of liquid, typically in combination with optical spectroscopy. Flow cytometry has enabled cell characterization even at a genomic level. A contrast mechanism in flow cytometry is fluorescence, externally administered or generated by genetic manipulation. To observe biological specimens in their most native condition, however, stain-free methods utilizing intrinsic properties of a specimen are preferred. Furthermore, the fluorescent labeling is not always a viable option especially for primary cells, and photobleaching and non-uniform binding of the labeling agents usually make it difficult to accurately quantify the target molecules.
Refractive index serves as a source of intrinsic contrast in a variety of imaging modalities including optical coherence tomography and light-scattering spectroscopy. At the same time, the refractive index can be related to the density of organic molecules, and its volume integral can provide the total amount of non-aqueous content in a cell or organelles. Variance and change in the refractive index of cells have been also linked to carcinogenic transformations. The refractive index of homogeneous bulk materials can be obtained with a critical angle refractometer measuring the critical angle of a specimen with respect to the other material with known refractive index. For thin layered materials, ellipsometry measuring depolarization of the incident light is known to be accurate. The refractive index of a non-homogeneous specimen such as biological cells, however, requires a more delicate treatment.
Tomographic phase microscopy (TPM) is a novel technique that has enabled 3-D mapping of refractive index in living cells. Recent developments in TPM promise stain-free monitoring of the physiological status of living cells at the sub-cellular level. Typically, the tomographic imaging of refractive index requires recording multiple images at varying angles of illumination on a specimen; therefore, existing methods had moving elements to change the illumination angle relative to the specimen that was stationary during the data acquisition, or alternatively rotated the specimen. As a result, the throughput of tomographic imaging is usually low, which has prevented TPM from being used for routine biological investigation.
The refractive index can be related to the speed of light wave inside a material. Therefore, wavefront distortion due to a specimen represents the total phase (time) delay of the light wave induced by the specimen. The wavefront distortion can be measured with a Shack-Hartman wavefront sensor, interferometry, or inline holography (also called propagation-based methods). Among these techniques, interferometry is particularly appropriate in the optical regime, where the light sources with a reasonably large coherence length are readily available. The phase delay is proportional to the specimen's optical path length, the integral of refractive index along the light propagation direction. Thus, as described herein the depth-resolved refractive index map may be acquired using a tomographic reconstruction algorithm in conjunction with the wavefront measurement. The tomographic refractive index measurement is typically performed with a collimated laser beam whose angle of incidence onto the sample is varied as in X-ray computer tomography. Alternatively, the refractive index map may be obtained with a spatially-incoherent beam and scanning the objective focus through the sample. In either approach, however, the sample has to be stationary while the illumination direction or the objective focus is varied, which limits the throughput of imaging.
Consequently, further improvements in tomographic phase microscopy are needed to improve the speed and accuracy of imaging systems.